Despite over 20 years of clinical use, IL-2 has not fulfilled expectations as a safe and effective form of tumour immunotherapy. Expression of the high affinity IL-2R&alpha; chain on regulatory T cells mitigates the anti-tumour immune response and its expression on vascular endothelium is responsible for life threatening complications such as diffuse capillary leak and pulmonary oedema. Here we describe the development of a recombinant fusion protein comprised of a cowpox virus encoded NKG2D binding protein (OMCP) and a mutated form of IL-2 with poor affinity for IL-2R&alpha;. This fusion protein (OMCP-mutIL-2) potently and selectively activates IL-2 signalling only on NKG2D-bearing cells, such as natural killer (NK) cells, without broadly activating IL-2R&alpha;-bearing cells. OMCP-mutIL-2 provides superior tumour control in several mouse models of malignancy and is not limited by mouse strain-specific variability of NK function. In addition, OMCP-mutIL-2 lacks the toxicity and vascular complications associated with parental wild-type IL-2.

f1: Generation of OMCP-mutIL-2.(a) Schematic structure of OMCP-mutIL-2. (b) Molecular mass of OMCP-mutIL-2 compared with mutIL-2 and wild-type IL-2 by Coomassie staining. wt IL-2, mutIL-2 and OMCP-mutIL-2 were produced in FreeStyle 293-F cells and purified from supernatants by Ni-NTA chromatography. The proteins have higher molecular mass due to glycosylation. The lower migrating band for mutIL-2 corresponds to unglycosylated protein. Based on differences in molecular mass all cytokines and construct were administered on a molar basis with 1 μl of 4.4 μM solution defined as 1000 IU equivalents (IUe) throughout the manuscript. This effectively allows for equimolar comparison between wt IL-2, mutIL-2 and OMCP-mutIL-2 despite different molecular mass. (c) Binding of various biotin-labelled cytokines and constructs to wild-type C57BL/6 splenic NK cells without (left panel) or with (middle panel) preincubation with OMCP. Binding of biotin-labelled cytokines and constructs to C57BL/6NKG2D−/− NK cells (right panel). As described in the methods spleen cells were co-cultured with equimolar concentration of biotinylated cytokines and fusion construct at 4 °C followed by labelling with phycoerythrin (PE) conjugated streptavidin and mixed fluorochrome-conjugated antibodies for defining of NK cells. Data were acquired flow cytometrically and is representative of three separate experiments with MFI comparison of PE-labelled constructs on NK cells (viable, NK1.1+, CD3−, CD19−). Data analysis performed by ANOVA for multiple comparisons and unpaired t-test for individual comparisons and shown as mean±s.e.m. All bar graphs represent mean±s.e.m. ns P>0.05; *P<0.05; black=saline; blue=wtIL-2, red=OMCP-mutIL-2, green=mutIL-2.

Mentions:
We designed an IL-2 fusion protein combining the high affinity NKG2D ligand OMCP with an IL-2 mutated to reduce IL-2Rα reactivity (mutIL-2). Our construct, termed OMCP-mutIL-2, consists of the 152 residue OMCP protein fused to the N-terminus of the 133 amino acid R38A/F42K mutant form of human IL-2 (mutIL-2) via a flexible 30 residue linker (Fig. 1a,b). We incubated equimolar concentration of biotinylated wild-type IL-2 (wtIL-2), mutIL-2 or OMCP-mutIL-2 with C57BL/6 splenocytes at 4 °C in vitro and compared binding flow cytometrically. The addition of the OMCP to mutIL-2 increased the retention of the fusion protein to NK cells compared with mutIL-2 or wtIL-2 (Fig. 1c left panel) as evidenced by significantly higher MFI. This increase in lymphocyte binding depended on functional and reactive NKG2D, as competitive preincubation of splenocytes with free monomeric OMCP eliminated enhanced binding of OMCP-mutIL-2 to NK cells (Fig. 1c middle panel). Consistent with this no increase in OMCP-mutIL-2 binding over mutIL-2 was evident in NK cells from C57BL/6NKG2D−/− mice (Fig. 1c right panel). No increased binding of OMCP-mutIL-2 over mutIL-2 was evident for either wild-type or C57BL/6NKG2D−/− B or T lymphocytes (Supplementary Fig. 1). Taken together our data demonstrate that a fusion protein consisting of a cytokine and an NKG2D ligand may have utility for targeting NKG2D expressing lymphocytes such as NK cells.

f1: Generation of OMCP-mutIL-2.(a) Schematic structure of OMCP-mutIL-2. (b) Molecular mass of OMCP-mutIL-2 compared with mutIL-2 and wild-type IL-2 by Coomassie staining. wt IL-2, mutIL-2 and OMCP-mutIL-2 were produced in FreeStyle 293-F cells and purified from supernatants by Ni-NTA chromatography. The proteins have higher molecular mass due to glycosylation. The lower migrating band for mutIL-2 corresponds to unglycosylated protein. Based on differences in molecular mass all cytokines and construct were administered on a molar basis with 1 μl of 4.4 μM solution defined as 1000 IU equivalents (IUe) throughout the manuscript. This effectively allows for equimolar comparison between wt IL-2, mutIL-2 and OMCP-mutIL-2 despite different molecular mass. (c) Binding of various biotin-labelled cytokines and constructs to wild-type C57BL/6 splenic NK cells without (left panel) or with (middle panel) preincubation with OMCP. Binding of biotin-labelled cytokines and constructs to C57BL/6NKG2D−/− NK cells (right panel). As described in the methods spleen cells were co-cultured with equimolar concentration of biotinylated cytokines and fusion construct at 4 °C followed by labelling with phycoerythrin (PE) conjugated streptavidin and mixed fluorochrome-conjugated antibodies for defining of NK cells. Data were acquired flow cytometrically and is representative of three separate experiments with MFI comparison of PE-labelled constructs on NK cells (viable, NK1.1+, CD3−, CD19−). Data analysis performed by ANOVA for multiple comparisons and unpaired t-test for individual comparisons and shown as mean±s.e.m. All bar graphs represent mean±s.e.m. ns P>0.05; *P<0.05; black=saline; blue=wtIL-2, red=OMCP-mutIL-2, green=mutIL-2.

Mentions:
We designed an IL-2 fusion protein combining the high affinity NKG2D ligand OMCP with an IL-2 mutated to reduce IL-2Rα reactivity (mutIL-2). Our construct, termed OMCP-mutIL-2, consists of the 152 residue OMCP protein fused to the N-terminus of the 133 amino acid R38A/F42K mutant form of human IL-2 (mutIL-2) via a flexible 30 residue linker (Fig. 1a,b). We incubated equimolar concentration of biotinylated wild-type IL-2 (wtIL-2), mutIL-2 or OMCP-mutIL-2 with C57BL/6 splenocytes at 4 °C in vitro and compared binding flow cytometrically. The addition of the OMCP to mutIL-2 increased the retention of the fusion protein to NK cells compared with mutIL-2 or wtIL-2 (Fig. 1c left panel) as evidenced by significantly higher MFI. This increase in lymphocyte binding depended on functional and reactive NKG2D, as competitive preincubation of splenocytes with free monomeric OMCP eliminated enhanced binding of OMCP-mutIL-2 to NK cells (Fig. 1c middle panel). Consistent with this no increase in OMCP-mutIL-2 binding over mutIL-2 was evident in NK cells from C57BL/6NKG2D−/− mice (Fig. 1c right panel). No increased binding of OMCP-mutIL-2 over mutIL-2 was evident for either wild-type or C57BL/6NKG2D−/− B or T lymphocytes (Supplementary Fig. 1). Taken together our data demonstrate that a fusion protein consisting of a cytokine and an NKG2D ligand may have utility for targeting NKG2D expressing lymphocytes such as NK cells.

Despite over 20 years of clinical use, IL-2 has not fulfilled expectations as a safe and effective form of tumour immunotherapy. Expression of the high affinity IL-2R&alpha; chain on regulatory T cells mitigates the anti-tumour immune response and its expression on vascular endothelium is responsible for life threatening complications such as diffuse capillary leak and pulmonary oedema. Here we describe the development of a recombinant fusion protein comprised of a cowpox virus encoded NKG2D binding protein (OMCP) and a mutated form of IL-2 with poor affinity for IL-2R&alpha;. This fusion protein (OMCP-mutIL-2) potently and selectively activates IL-2 signalling only on NKG2D-bearing cells, such as natural killer (NK) cells, without broadly activating IL-2R&alpha;-bearing cells. OMCP-mutIL-2 provides superior tumour control in several mouse models of malignancy and is not limited by mouse strain-specific variability of NK function. In addition, OMCP-mutIL-2 lacks the toxicity and vascular complications associated with parental wild-type IL-2.